Induced pluripotent stem (iPS) cells are a type of pluripotent stem cells resembling embryonic stem (ES) cells and can be derived from adult somatic cells by forced expression of certain genes. Both human ES and iPS cells are able to give rise to all the fully differentiated tissues and may provide an ideal source for the production of heart cells for cell-based therapies. Although human iPS cell-derived cardiomyocytes are functionally comparable to those derived from human ES cells in vitro, their functionality has not been compared in a physiologically relevant context. This proposal will test the hypothesis that human iPS cell- derived heart cells are the functional equivalent of their human ES cell-derived counterparts and thus provide an autologous cell source for cardiac repair and cardiovascular research. A mixture of cell types enriched for heart cells can be isolated from human ES and iPS cells in culture, but in order to create functional heart tissue and obtain reproducible results in cell-based therapies, it is critically important to isolate pure populations of cardiac cells that will engraft and promote cardiac repair. Ventricular cardiomyocytes (VCM) are the major cell type injured in patient's hearts, resulting in reduced cardiac function. While implanted VCM may be the optimal mature cell type to readily couple electrically with host VCM, our recent discovery of ISL1+ cardiovascular progenitor cells (ICPC) may represent a better source for cell-based therapies as these are highly proliferative cells that have the capacity to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells, all of which are required for cardiac repair. By combining bacterial artificial chromosomal (BAC) recombineering technology with human ES cell engineering, the Qyang laboratory has established human ES and iPS cell lines that harbor enhanced green fluorescent protein (eGFP) and puromycin-resistance (Puro) dual reporters controlled by the endogenous myosin light chain 2V (MLC2V) promoter, which is VCM specific. Such a strategy has enabled the Qyang laboratory to isolate highly purified VCM based on both GFP expression and Puro-resistance. We will use this same technology to derive pure ICPC by establishing human ES and iPS cell lines that harbor GFP and Puro dual reporters controlled by the endogenous ISL1 promoter. As a proof of principle study to generate patient-specific VCM and ICPC for cell-based therapies, the Qyang laboratory proposes to isolate and characterize VCM and ICPC heart cells generated from human iPS cells using traditional 2D cultures and a recently established, physiologically relevant 3D "cardiac organoid chamber" system in vitro as well as cardiac repair models in vivo. For cardiac repair in vivo, we will employ a recently developed "cell sheet engineering" technology to establish engineered heart tissues. For all experiments, heart cells derived from NIH-approved human ES cells will be used as controls. PUBLIC HEALTH RELEVANCE: This research will lead to the production of pure, well-characterized ISL1+ cardiovascular progenitor cells and ventricular cardiomyocytes as an abundant renewable cell source for cardiac repair. Development of engineered cardiac tissues using human ES and iPS cell cultures will provide exciting opportunities for high- throughput, patient-specific evaluation of cardiac medicines and cell-based therapies for heart failure.